CD81 is a tetraspanin with several immunoregulatory functions. On T cells antibody to CD81 activates LFA-1 and can provide co-stimulatory signals with TCR/CD3 to promote T cell activation. Antibody to CD81 can also block early T cell development. CD81-/- mice have impaired Th2 responses and are resistant to allergen induced airway hyperreactivity. CD81 is a receptor for the hepatitis C virus (HCV) envelope protein E2, and binding of E2 to CD81 induces effects similar to those caused by anti-CD81 antibodies. The mechanism by which CD81 regulates T cell activity is unknown. It was recently discovered that on T cells CD81 is physically associated with a novel Ig-superfamily member called PGRL. PGRL may regulate prostaglandin (PG) responses on T cells. In broad terms this proposal seeks evidence of a connection between specific PGs, CD81 and the regulation of T cell adhesion and motility via LFA-I. Specifically this project will: 1) Identify the CD81-mediated signals that cause LFA-1 activation on T cells. The role of palmitoylation and the recruitment of a specific scaffold protein involved in signal transduction will be studied. A novel LFA-1 avidity assay using flow cytometry and ICAM-coated fluorescent microspheres will be used to identify signaling intermediates between CD81 and LFA-1. 2) Define the structural and molecular features of CD81-PGRL complexes. This aim will employ CD81 and PGRL mutagenesis, and biochemical techniques including MALDI-TOF MS to test the hypothesis that CD81 serves as a transmembrane adapter protein linking PGRL with an intracellular signaling protein. 3) Determine whether CD81 and/or PGRL interact with or regulate the activity of EP3. The affinity of EP3 will be tested in the presence or absence of antibody to CD81 or PGRL and tested under conditions which control the co-expression of PGRL and CD81 or in the presence of soluble PGRL. The coordinated effect of CD81, PGRL and EP3 on T cell integrin activity measured by adhesion to or motility on ICAMs.